Apomixis in Achnanthes (Bacillariophyceae); development of a model system for diatom reproductive biology

The availability of extensive experimental data and remarkable intra- and interspecific variation in breeding behaviour make Achnanthes Bory sensu stricto an especially good model for studying the reproductive and population biology of pennate diatoms. In most Achnanthes species studied, auxospore formation is accompanied by biparental sexual reproduction, but we found uniparental auxosporulation in Achnanthes cf. subsessilis. Auxosporulation appears to be apomictic and follows contraction of the contents of unpaired cells and then a mitotic division, which is normally acytokinetic: one nucleus aborts before the cell develops into an auxospore. Rarely, both daughter nuclei survive and cytokinesis produces two auxospores (two auxospores per mother cell is highly unusual in pennate diatoms); one may abort. Expansion of auxospores is not accompanied by deposition of a transverse perizonium, but a longitudinal perizonium is produced and consists of a wide central strip (structurally similar to the araphid valve) and at least one narrow lateral strip. This newly discovered asexual lineage in Achnanthes is discussed in relation to other reproductive systems found in the genus, and also in relation to the ‘sex clock’ hypothesis concerning the adaptive significance of the diatom life cycle. Brief information on chloroplast division and nuclear dynamics over the cell cycle is also presented.     

SEXUAL REPRODUCTION IN NAVICULA CRYPTOCEPHALA (BACILLARIOPHYCEAE)1

Homothallic sexual reproduction and auxosporulation were studied in monoclonal cultures and seminatural populations of the freshwater epipelic diatom Navicula cryptocephala Kütz. Gametangia paired via the girdle, one gamete was formed per gametangium (and hence one zygote per pair of gametangia), and gamete fusion took place without the formation of any copulation envelope or copulation canal. Superfluous nuclei from meiosis survived unusually long, so that gametes and young zygotes were probably functionally polyploid; later, all but two haploid nuclei degenerated. Expanded auxospores had a swollen center, but during formation of the initial valves, the auxospore contracted away from the perizonium to produce linear‐lanceolate valves. The pattern of reproductive behavior found in N. cryptocephala can be classified as type IIA2a auxosporulation in Geitler's system. The same type of zygote and auxospore formation seen in clonal cultures was observed in seminatural material from four lakes in Scotland and the Czech Republic. Variation in nuclear structure and auxosporulation in the N. cryptocephala species complex is discussed, as is the evolution of type II auxosporulation (one zygote per pair of gametangia) from type I auxosporulation (two zygotes per pair). The penalty of smaller numbers of zygote produced in type II may be outweighed by formation of larger auxospores (prolonging the vegetative phase) or more vigorous auxospores. The variation present among members of the N. cryptocephala complex indicates that biogeographical analyses based on use of the name N. cryptocephala, as performed recently to support the ubiquity hypothesis of protist distributions, are almost meaningless.     

SCALY INCUNABULA,AUXOSPORE DEVELOPMENT,AND GIRDLE POLYMORPHISM IN SELLAPHORA MARVANII SP. NOV. (BACILLARIOPHYCEAE)1

Uniparental auxosporulation was observed in a monoclonal culture of a Sellaphora clone isolated from the epipelon of a fishpond in the Czech Republic. The cox1 sequence for the clone confirmed that it belonged to the Sellaphora pupula–bacillum species complex but showed significant differences from all previously characterized Sellaphora species, and it is therefore described as S. marvanii sp. nov. Protoplast, valve, and girdle structure resembled those of other Sellaphora species, but a novel finding for all diatoms was a change in girdle structure during the life cycle: the most advalvar girdle band (valvocopula) bore a single line of pores in enlarged postauxospore cells but was entirely plain in small cells and gametangia. The young auxospores were covered by incunabula containing large, delicate, ± circular scales, resembling those of centric diatom auxospores; similar scales have been reported in a few other raphid diatoms (Pseudo‐nitzschia multiseries, Diploneis sp.) but contrast with the strip incunabula of some Nitzschia and Pinnularia and the helmet‐like caps of Neidium. The scales persisted during auxospore expansion, mostly as two caps over the auxospore poles. The transverse perizonium comprised a very wide, closed primary band, flanked by numerous secondary bands whose open ends were strongly incurved toward the center. Initial valves were differentiated from their immediate descendants by the very strong external demarcation of the raphe sternum, irregular shape, and curved transapical profile.     

AUXOSPORE FORMATION BY THE SILICA‐SINKING,OCEANIC DIATOM FRAGILARIOPSIS KERGUELENSIS (BACILLARIOPHYCEAE)1

Size restoration by the auxospore that develops from the zygote is a crucial stage in diatom life cycles. However, information on sexual events in pelagic diatom species is very limited. We report for the first time auxospore formation by the pennate diatom Fragilariopsis kerguelensis (O'Hara) Hustedt during an iron‐induced bloom in the Southern Ocean (EIFEX, European Iron Fertilization EXperiment). Auxospores of F. kerguelensis resembled those described for Pseudo‐nitzschia species. The auxospore was characterized by an outer coating, the perizonium; two caps, one at each distal end; and four chloroplasts, one at each end and two in the central part. Different stages of auxospore elongation were recorded, with a length of 24–91 μm, but only the largest auxospores contained the initial cell, whose apical axis ranged between 76 and 90 μm. Gametangial cell walls were often attached to the auxospores and ranged from 10 to 31 μm in length. Auxospore abundances were consistently higher in the fertilized patch, where an increase in the F. kerguelensis population was observed, as compared with surrounding waters.     

AUTOGAMOUS AUXOSPORULATION IN PINNULARIA NODOSA (BACILLARIOPHYCEAE)1

Auxosporulation of the freshwater epipelic diatom Pinnularia nodosa (Ehrenb.) W. Sm. was studied in a clonal culture. Interphase cells possessed two chloroplasts with invaginated pyrenoids. The nucleus contained a single small body of heterochromatin at one end, also visible during most of meiotic prophase. During auxosporulation, induced by transfer of stationary‐phase cells to fresh medium and suppressed by high nitrogen (N), an unpaired mother cell produced a single auxospore. Although meiosis II and nuclear fusion were not observed, indirect evidence indicated that auxosporulation was autogamous (rarely reported in pennate diatoms), rather than apomictic; paedogamy was excluded. The protoplast produced after meiosis either (1) matured into a “pseudozygote,” via an asymmetrical contraction after meiosis I to form a single spherical cell at one end of the mother cell (pathway 1); or (2) constricted into two spherical cells (pathway 2). In pathway 2, the “pseudogametes” never fused and only one or none developed into a pseudozygote and then into an auxospore. Pathway 2 could be suppressed by continuous light. During metamorphosis of the spherical pseudozygote into an elongate young auxospore, a complete covering of thin siliceous incunabular strips was formed, separate from the organic wall formed around the pseudozygote when first formed and from the perizonium. Mature auxospores produced via pathway 2 had 60% of the volume as those produced via pathway 1 and had smaller chloroplasts (through loss of fragments during protoplast cleavage), but they achieved exactly the same lengths, suggesting that absolute length is monitored during expansion.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.     

Silicification of auxospores in the araphid diatom Tabularia fasciculata (Bacillariophyta)

We report on auxospore wall structure and development in the araphid pennate diatom Tabularia fasciculata. Similar to most other pennates, these auxospores showed a typical bidirectional elongation, but unexpectedly bore no transverse perizonium, and with no detectable silicon during much of their expansion. Energy dispersive X-ray spectroscopy (EDS) analyses segregated auxospores into two types: (1) those containing no detectable silicon and (2) those with measureable amounts. Both types were of similar size. Silica precipitation began throughout the auxospore at or near maximal length, but initially was detectable in isolated regions throughout the structure. Following this initial condition, silicon was consistently detectable throughout auxospores of comparable size and corresponded to deposition of longitudinal perizonium (visible through the thin organic outer layer of the wall in some auxospores), followed by the deposition of the initial valves. Our results raise the question as to how the tubular shape of bidirectionally expanding auxospores up to ∼90 μm long is maintained in the absence of transverse siliceous elements restricting isodiametric expansion of the cell, which are present in all other known pennate auxospores and all but one other diatom. Our study is the first to systematically examine mineral elements of the auxospore wall analytically.     

TAXONOMY,LIFE CYCLE,AND AUXOSPORULATION OF NITZSCHIA FONTICOLA (BACILLARIOPHYTA)1

Nitzschia fonticola (Grunow) Grunow is a member of Nitzschia sect. Lanceolatae, a group of taxonomically intractable but ecologically important and widespread diatoms. We investigated the morphology and life cycle in three clones of N. fonticola and all exhibited reduced sexuality, with pedogamous production of auxospores in unpaired gametangia. The auxospores of all clones contained tangles of striplike elements that lay outside the perizonium and were distinct from it in structure and ontogeny. We introduce a new term, incunabula, to refer to such components of the auxospore wall. Semicryptic variation was detected: one clone differed from the other two in valve size and shape, stria density, and fibula density, as well as its nuclear large subunit ribosomal DNA (LSU rDNA) sequence. The implications of reduced sexuality for the taxonomy of sect. Lanceolatae are discussed. A lectotype is designated for N. fonticola from among original material of Grunow, and the application of the name is clarified further by designating illustrations and the LSU sequence AM182191 from one of our clones as epitypes.     

Female gametogenesis and female gamete germination in the anisogamous green alga Codium fragile subsp. novae‐zelandiae (Bryopsidophyceae,Chlorophyta)

Female gametogenesis was studied in the dioecious siphonous green alga Codium fragile subsp. novae‐zelandiae (J. Agardh) P. C. Silva using light and electron microscopy. Early during gametogenesis the protoplasm was uniform; then it separated in portions, while fusiform chloroplasts and nuclei increased in numbers. Some features of the nuclear divisions were similar to those of other Bryopsidophyceae. They were acentric and semi‐open. Pairs of parallel electron‐dense lines resembling synaptonemic complexes were observed in several prophase nuclei indicating meioses. In metaphase the nuclear envelope showed polar fenestrae from which the spindle emerged. No spindle microtubule nucleating material was visible and chromosome kinetochores were evident. Mature female gametes were pyriform with a hyaline anterior end from which the two flagella emerged. Mature gametes had a spherical nucleus surrounded by a mitochondrion and numerous discoid chloroplasts. Female gametes germinated parthenogenetically in culture and also inside gametangia, involving loss of flagella, rounding and lengthening of cells, multiplication of chloroplasts with well developed thylakoid systems, vacuolization and synthesis of a fibrillar cell wall.     

LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS OF PREFERENTIAL DESTRUCTION OF CHLOROPLAST AND MITOCHONDRIAL DNA AT EARLY MALE GAMETOGENESIS OF THE ANISOGAMOUS GREEN ALGA DERBESIA TENUISSIMA (CHLOROPHYTA)1

Gametogenesis in male and female gametophytes was studied by light microscopy and EM in the dioecious multinucleate green alga Derbesia tenuissima (Moris & De Notaris) P. Crouan & H. Crouan, where male and female gametes differ in size. Gametogenesis was divided into five stages: 32 h (stage 1), 24 h (stage 2), 16 h (stage 3), 8 h (stage 4), and 0.5 h (stage 5) before gamete release. At stage 1, the first sign of gametogenesis observed was the aggregation of gametophyte protoplasm to form putative gametangia. At stage 2, gametangia were separated from the vegetative protoplasm of gametophytes. Morphological changes of nuclei and organelles occurred at this early stage of male gametogenesis, and organelle DNA degenerated. At stage 3, male organelle DNA had completely degenerated, whereas in female gametangia, organelle DNA continued to exist in both chloroplasts and mitochondria. Gametogenesis was almost completed at stage 4 and fully at stage 5. Small male gametes had a DNA‐containing nucleus and a large mitochondrion and one or several degenerated chloroplasts. The mitochondria and plastids were devoid of DNA. The large female gametes had a nucleus and multiple organelles, all of which contained their own DNA. Thus, degeneration of chloroplast DNA along with morphological changes of organelles occurred at male gametogenesis in anisogamous green algae (Bryopsis and D. tenuissima), in contrast with previous studies in isogamous green algae (Chlamydomonas, Acetabularia caliculus, and Dictyosphaeria cavernosa) in which degeneration of chloroplast DNA occurred after zygote formation.     

MATING SYSTEM,SEXUAL REPRODUCTION,AND AUXOSPORULATION IN THE ANOMALOUS RAPHID DIATOM EUNOTIA (BACILLARIOPHYTA)1

The diatom genus Eunotia is unusual among raphid diatoms in having a raphe system consisting of two short slits that are not integrated into the primary pattern center. This and other characteristics, particularly the presence of rimoportulae, are consistent with the hypothesis that Eunotia is a basal lineage within the raphid group. We studied auxosporulation in E. bilunaris (Ehrenberg) Mills and E. tropica Hustedt for comparison with other raphid pennate diatoms and with araphid pennates; E. bilunaris was studied in parental and F1 generations. Like araphid pennates, E. bilunaris and E. tropica are heterothallic. Clones of the same mating type did not interact sexually, and intraclonal sexual reproduction was absent or very rare. Clones retained the same sex throughout the life cycle, as shown by experiments using abrupt size reduction to produce clones of similar age but different size and using subclones derived from a single initial cell within six mitotic generations. Unlike in araphid pennate diatoms, in the Eunotia species the gametes are not visibly or behaviorally differentiated. Gametogenesis is merogenous, because the gametangium formed a supernumerary cell as well as a single gametic cell, both undergoing meiosis II to form a surviving functional nucleus and a nucleus that quickly degenerated. Plasmogamy is via papillae that grew out toward each other from the ends of the gametangia to create a copulation canal. After plasmogamy, the gametes moves bodily into the copulation canal, producing an elongate zygote, which expands to form a curved sausage‐like auxospore.     

The systematics of Seminavis (Bacillariophyta) : the lost identities of Amphora angusta,A. ventricosa and A. macilenta

Amphora angusta Gregory, Amphora ventricosa Gregory and Amphora macilenta Gregory have been misunderstood for the last 140 years. Gregory described these species from sublittoral sediments off the west coast of Scotland. Although the illustrations were excellent for their time (1857), it is impossible to be confident of identification using Gregory's paper, and subsequent authors have created such confusion that records of these taxa must be regarded as untrustworthy unless backed by photographic evidence. In this paper, the first to describe in detail the recently established genus Seminavis, we show that A. angusta is in fact a species of Amphora Ehrenberg, whereas A. ventricosa sensu Gregory represents two independent species of the genus Seminavis D. G. Mann, namely Seminavis ventricosa (Gregory) Garcia-Baptista (non S. ventricosa sensu Garcia-Baptista 1993) and Seminavis arranensis Danielidis & D. G. Mann, sp. nov. The form usually known as A. ventricosa is neither of these and requires a new name, Seminavis robusta Danielidis & D. G. Mann, sp. nov. The long forgotten and misclassified species A. macilenta Gregory is shown to be yet another Seminavis species, requiring a new combination as S. macilenta (Gregory) Danielidis & D. G. Mann, comb. nov.; contrary to previous claims, it is separate from A. ergadensis Gregory, which is a true Amphora. Valve and girdle ultrastructure, plastid arrangement and auxosporulation are described for Seminavis and resemble those in the Naviculaceae sensu stricto.     

SEXUAL REPRODUCTION OF PERIDINIUM GATUNENSE (DINOPHYCEAE)1

Sexual reproduction was induced in the dinoflagellate Peridinium gatunense Nygaard when exponentially growing cells were inoculated into nitrogen deficient medium. Small thecate cells produced by division of vegetative cells then acted as gametes. Thecae of fusing gametes broke in the girdle region and were lost. Zygotes thus formed remained motile 3–5 days during which time they enlarged slightly with the newly formed theca becoming warty. Three to 5 days following plasmogamy the zygote became nonmotile, the protoplast contracted, and the cell wall thickened. Hypnozygotes with 4 nuclei were observed ca. 10–12 h following formation. Meiosis was inferred. Hypnozygotes germinated within 12 h of formation producing 2 vegetative cells which divided within a 24 h period. Attempts to induce sexual reproduction by inoculation of cells into media deficient in a number of basic elements other than N were unsuccessful.     

Observations on girdle bands during cell division in the diatom Stephanodiscus

The fate of old girdle bands and the formation of new bands in the centric diatom Stephanodiscus are discussed. The use of scanning electron microscopy is a great aid in such studies since it enables observation of complete cells and determination of the position and overlap of girdle bands. It appears that both parent girdle bands are lost during cell division and two new bands formed around each daughter cell.     

Auxospore formation inLicmophora (Bacillariophyta)

InLicmophora gracilis var.anglica two auxospores are produced per pair of mother-cells, through the allogamic fusion of migratory and stationary gametes. Both active gametes are produced from the same mother-cell and hence both zygotes are formed in the other mother-cell. Pairing can occur between two stalked cells, or between a stalked cell and a detached cell; in the latter case the migratory gametes derive from the detached cell. The auxospores expand parallel to one another and to the apical axis of the donor mother-cell. Behavioural anisogamy of this kind, which may be termed thecis-type, seems to be characteristic of most araphid pennates and contrasts with thetrans-type exhibited byCymbella, Gomphonema and some other raphid taxa, where each mother-cell produces one migratory and one stationary gamete.     

Preferential digestion of chloroplast DNA in male gametangia during the late stage of gametogenesis in the anisogamous algaBryopsis maxima

Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).     

Morphological investigations of the frustule, perizonium and initial valves of the freshwater diatom Achnanthes crenulata Grunow (Bacillariophyceae)

The present study clarifies the fine structure of the vegetative frustules, initial valves and perizonium of Achnanthes crenulata Grunow. The valves of the vegetative cell are distinctly linear‐lanceolate with an undulate margin. The valve face is quite flat and in girdle view is smoothly curved as in species of Gephyria (Bacillariophyceae). However, the valve face of the initial cells is slightly rounded and does not have an undulate margin. Furthermore, the rapheless sternum is centrally positioned along the apical axis of the araphid initial valve. As this taxon develops from auxospore to initial valve, it forms only longitudinal perizonial bands; no transverse bands arise. The perizonium consists of three silicified bands: one large, central longitudinal plate and two bands that underlie this plate; these two bands are either open or closed. This taxon has several conspicuous structures compared to other marine species of Achnanthes, but the structure of the perizonium supports the position of A. crenulata within Achnanthes sensu stricto.     

Auxospore Structure, Initial Valves and the Development of Populations of Stephanodiscus in Farmoor Reservoir

A population of the planktonic diatom Stephanodiscus growingin Farmoor Reservoir has been investigated with regard to auxosporeformation and the subsequent change in cell size. This is thefirst account of auxospores in Stephanodiscus. The auxosporewall is believed to be organic without any siliceous components.Germination results in two large hemispherical initial valveswhich are rather crudely formed and lack the ring of spinescharacteristic of the vegetative cells which are figured forcomparison. The division of the initial cell to form hemisphericalcells is recorded. These then form a normal vegetative celland a hemispherical cell. The hemisperhical cells acted as markersin the natural population and their longevity was determined.The natural populations did not exhibit a continuous decreasein cell diameter, at times remaining almost constant and atother times decreasing. The origin and ratio of occurrence ofbiconcave, biconvex and concavo-convex cells in the populationis discussed. Stephanodiscus, diatom, auxospore, diatom cell size     

Electron microscopical observations onPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the centric diatomThalassiosira punctigera

Summary Ultrastructural observations revealed details of the infection process and the fine structure ofPseudaphelidium drebesii Schweikert and Schnepf, a parasite of the marine centric diatomThalassiosira punctigera (Castracane) Hasle. After attachment and encystment of the zoospore an intracellular infection tube is generated. This structure is everted between the overlap of the girdle bands or the gap between the valve and the girdle band of the host diatom. The bulk of thePseudaphelidium protoplast enters the silica shell of the diatom via the infection tube but does not pierce the host plasma membrane. Parts of the host cytoplasm are phagocytized with microfilament involvement.Pseudaphelidium drebesii is characterized by unusual microbodies containing tubular inclusions and by closed mitosis with a perinuclear spindle. The taxonomic position ofP. drebesii is discussed.